The long term goal of this proposal is to understand the role salmolysin, a cytolysin identified in Salmonella, plays in Salmonella virulence. The sly gene encodes the salmolysin protein which possesses both hemolytic and cytolytic activity. The sly gene has been cloned and sequenced, and salmolysin has been partially purified and biochemically characterized. The sly gene is conserved among Salmonella serotypes examined. Inactivation of the sly gene profoundly attenuates Salmonella typhimurium for virulence by the intraperitoneal, oral, and intravenous routes of infection. Salmolysin mutants (sly-) are unable to survive and replicate in murine macrophages in vitro. The central hypothesis of this proposal is that sly is one of the major genes involved in the characteristic ability of Salmonella to survive and replicate within the cells of the reticuloendothelial system. In this proposal, the survival and replication of a sly mutant of Salmonella typhimurium will be examined in murine macrophages from different sources. To determine the biological role of salmolysin in the ability of Salmonella to survive and replicate in macrophages, various macrophage functions will be assayed following infection with wild type and a sly mutant of Salmonella. The regulatory gene(s) that control the expression of salmolysin will be identified by transposon mutagenesis of the Salmonella typhimurium chromosome. Reporter gene fusions will be used to determine if salmolysin gene expression is induced in the presence of macrophages. The following Specific Aims are proposed: 1) to determine how salmolysin is regulated using chromosomal reporter gene fusions and transposon mutagenesis. 2) to determine the cellular location of salmolysin. 3) to identify macrophage bactericidal functions which may be altered in the presence or absence of a functional sly gene. 4) to determine the expression of the sly gene in macrophages using reporter gene fusions.